Medical use of matrix metalloproteinase inhibitors for inhibiting tissue contraction

ABSTRACT

The use of an MMP inhibitor, especially a collagenase inhibitor, in the manufacture of a medicament for the treatment of a natural or artificial tissue comprising extracellular matrix components to inhibit contraction of the tissue and methods for the treatment of tissue comprising extracellular matrix components to inhibit contraction.

[0001] The present invention relates to matrix metalloproteinase (MMP)inhibitors, especially collagenase inhibitors, and to their use in themanufacture of medicaments.

[0002] There are many different types of collagen found in the body andthey, together with other extracellular matrix components, for example,elastin, gelatin, proteoglycan and fibronectin, make up a largeproportion of the body's extracellular tissue. Matrix metalloproteinases(MMPs) are enzymes that are involved in the degradation and denaturationof extracellular matrix components. Collagenases, for example, arematrix metalloproteinases that degrade or denature collagen.

[0003] A large number of different collagenases are known to exist.These include interstitial collagenases, type IV-specific collagenasesand collagenolytic proteinases. Collagenases are generally specific forcollagens which, in their full triple helix structure, are extremelyresistant to other enzymes.

[0004] Other MMPs are involved in the degradation and denaturing ofother extracellular matrix components, for example, elastin, gelatin andproteoglycan. Some MMPs are able to degrade or denature severaldifferent types of collagen and also other extracellular matrixcomponents. For example, stromelysin degrades type IV collagen, which isfound in basement membrane, and also has an effect on otherextracellular matrix components such as elastin, fibronectin andcartilage proteoglycans.

[0005] There is a classification system for MMPs, see Nagase et al 1992.For example, MMP1 is a collagenase that is sometimes called“collagenase”, MMP2 is a 72kD gelatinase, MMP3 is stromelysin and MMP9is a 92kD gelatinase. The official designations are used herein.

[0006] Collagenases have been implicated in a number of diseases, forexample, rheumatoid arthritis [Mullins, D. E. et al 1983], periodontaldisease and epidermolysis bullosa, and it has been proposed to use MMPinhibitors in the treatment of such conditions.

[0007] U.S. Pat. Nos. 5,183,900, 5,189,-178 and 5,114,953 describe thesynthesis ofN-[2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophanmethylamide, also known as GM6001, Galardin or Galardin-MPI (tradenames), and other MMP inhibitors, and their use in the prevention andtreatment of corneal ulceration. Treatment of corneal ulcers withpeptide hydroxamic acid inhibitors has been found to assist in thehealing of those ulcers. Further details of such uses are given inSchultz et al 1992.

[0008] Also described in the above-mentioned US patent specifications isthe use of collagenase inhibitors in situations where bacterial enzymesmay be detrimental to tissue, for example, in bacterial ulceration.

[0009] Other collagenase inhibitors based on hydroxamic acid aredisclosed in WO 90/05716, WO 90/05719 and WO 92/13831. Such collagenaseinhibitors are disclosed as being used in the management of diseaseinvolving tissue degradation, particularly disease involving collagenbreakdown, and/or the promotion of wound healing.

[0010] Other synthetic MMP inhibitors and in particular collagenaseinhibitors that have been developed include those-described inEP-A-126,974 and EP-A-159,396 and in U.S. Pat. Nos. 4,599,361 and4,743,587.

[0011] One inhibitor undergoing clinical trials is BB-94 also known asBatimastat (British Bio-technology Ltd.). Potential uses of BB-94 forthe control of cancer metatasis are described in EP-A-276436. It hasbeen proposed to use an oral formulation of BB-94 in the treatment ofbone cancer.

[0012] The present invention is concerned with the contraction oftissues, for example, scars. Contraction of tissues comprisingextracellular matrix components, especially of collagen-comprisingtissues, may occur in connection with many different pathologicalconditions and with surgical or cosmetic procedures. Contracture, forexample, of scars, may cause physical problems, which may lead to theneed for medical treatment, or it may cause problems of a purelycosmetic nature.

[0013] It has been proposed that contraction is cell-mediated and anumber of studies have suggested possible mechanisms for cell mediatedcollagen contraction [Gabbiani et al. 1972, Ehrlich & Rajaratnam 1990].Investigations have been made into the role, if any, played by MMPs inthe process of contracture. However, according to one proposition, MMPsare not produced during contraction [Schor et al. 1980]. According toanother proposition, MMPs are produced during lattice contraction butare not implicated in the contractile process [Nakagawa et al. 1989,Mauch et al. 1989, Lambert 1992]. Instead, it has been proposed thatcontraction is dependent upon the extracellular lattice cell number,upon there being an intact actin cytoskeleton, and upon attachment ofthe cells to the extracellular matrix.

[0014] The present invention is based on the surprising observationthat, during experiments on in vitro models of scar contraction,collagen (the main component of scar tissue) appears to be invaded andpermanently remodelled by fibroblasts and that such invasion andremodelling is inhibited by collagenase inhibitors. The remodellinggenerally appears as contraction of the collagen, which contraction isinhibited by inhibition of collagenase. Furthermore, inhibition of otherMMPs also results in inhibition of contraction. The observation thatcontraction of the tissue involves MMPs is particularly surprising sinceprevious investigations have shown that MMPs are not produced duringcontraction while other investigations have indicated that MMPs areproduced but are not involved in the contractile process (see above).

[0015] The present invention provides the use of an MMP inhibitor in themanufacture of a medicament for the treatment or prophylaxis of anatural or artificial tissue comprising extracellular matrix componentsto inhibit, i.e. restrict, hinder or prevent, contraction of the tissue,especially contraction resulting from a pathological condition or fromsurgical or cosmetic treatment.

[0016] The present invention also provides a method for the inhibitionin vivo or in vitro of contraction of a natural or artificial tissuecomprising extracellular matrix components, which comprisesadministering an MMP inhibitor to the tissue during and/or after itsformation. When the tissue is in vivo, a therapeutically effectiveamount of the MMP inhibitor should be administered.

[0017] The present invention especially provides the use of acollagenase inhibitor in the manufacture of a medicament for thetreatment or prophylaxis of tissue comprising collagen to inhibitcontraction of the tissue resulting from contraction of the collagen.

[0018] The present invention further especially provides a method forthe inhibition of contraction of tissue comprising collagen, resultingfrom contraction of the collagen, which comprises administering atherapeutically effective amount of a collagenase inhibitor to thetissue.

[0019] The methods of the present invention may be used for medical orcosmetic treatment.

[0020] The invention provides a method for the inhibition, for cosmeticreasons, of disfigurement caused by contraction of tissue comprisingextracellular matrix components, which method comprises administering amatrix metalloproteinase inhibitor to the tissue.

[0021] Cosmetic treatments, such as chemical or physical dermalabrasion, used as anti-ageing treatments, cause trauma to the skin. Useof MMP inhibitors during the healing process which occurs after theinitial abrasion is a cosmetic use of MMP inhibitors according to thepresent invention.

[0022] The present invention also provides the use of an MMP inhibitorto inhibit, i.e. restrict, hinder or prevent, invasion by cells,especially fibroblasts, into tissue comprising an extracellular matrixand/or migration by cells, especially fibroblasts, in or through tissuecomprising an extracellular matrix.

[0023] The term “MMP inhibitor” is used herein to denote any substancethat is capable of inhibiting, i.e. restricting, hindering orpreventing, the action of an MMP. The term “collagenase inhibitor” isused herein to denote any substance that is capable of inhibiting, i.e.restricting, hindering or preventing, the action of a collagenase. Acollagenase inhibitor may be specific for one particular collagenase ormay inhibit several different collagenases. A collagenase inhibitor mayalso inhibit other MMPs, in which case it may also be defined as an MMPinhibitor.

[0024] The term “inhibitor” as used herein includes agents that actindirectly by inhibiting the production of the relevant enzyme, forexample an antisense molecule, as well as agents that act directly byinhibiting the enzyme activity of the relevant enzyme, such as, forexample, a conventional inhibitor.

[0025] An MMP, e.g. collagenase, inhibitor may be naturally-occurring orsynthetic. An MMP, e.g. collagenase, inhibitor may be an anti-MMP, e.g.anti-collagenase, antibody, either polyclonal or monoclonal. Theinhibitory activity of a putative MMP inhibitor may be assessed by anymethod suitable for determining inhibitory activity of a compound withrespect to an enzyme. Such methods are described in standard textbooksof biochemistry. A more detailed description of MMP, e.g. collagenase,inhibitors is given below.

[0026] Collagen is the major component of scar and other contractedtissue and as such is the most important structural component toconsider. Nevertheless, scar and other contracted tissue also comprisesother structural components, especially other extracellular matrixcomponents, for example, elastin, which may also contribute tocontraction of the tissue. MMPs are involved in the synthesis anddegradation of such components. In general, a collagenase inhibitor isused as the MMP inhibitor in accordance with the present invention but,it may be appropriate to use instead an inhibitor of an MMP other than acollagenase. It may be particularly advantageous to use a combination ofa collagenase inhibitor and one or more inhibitors of other MMPs or touse an inhibitor which inhibits both a collagenase and at least one ormore other MMPs.

[0027] The mechanism and control of contraction of tissues comprisingextracellular matrix components, for example, collagen-comprisingtissues, is still poorly understood. Some degree of contraction appearsto be part of the healing process, but the trigger for contraction isnot known. The involvement of MMPs in contraction of tissues, forexample, scar tissue, and the utility of MMP inhibitors according to thepresent invention in the inhibition, i.e. prevention, restriction andhindering, of contraction has been confirmed by the followingexperimental data:

[0028] When fibroblasts in an in vitro collagen gel model of scarcontraction are subjected to antiproliferative agents after contractionhas occurred, there is no significant expansion of the collagen gel,that is to say, no relaxation of the contraction, even when thefibroblast cells have been killed and the supporting cytoskeleton of thecells removed. The remodelling of collagen leading to contractiontherefore appears to result from activity of one or more enzymic systemsof the fibroblasts.

[0029] Fibroblasts involved in the contraction of collagen producegreater amounts of matrix metalloproteinase mRNAs and proteins than docontrol fibroblasts. This is associated with cellular invasion of thecollagen. Invasion of the collagen and contraction are inhibited by theuse of inhibitors specific to those MMPs coded for by the mRNAs whichare present at higher levels in cells involved in contraction than incontrol cells:

[0030] Quantitative competitive reverse transcriptase polymerase chainreaction (QCRT-PCR) technique [Tarnuzzer & Schultz 1994] was used tostudy the levels of MMP mRNA produced by human ocular fibroblasts incollagen Type I lattices undergoing contraction in comparison with humanocular fibroblasts in monolayer cultures. It was found that in thecollagen lattices the fibroblasts produced more mRNA for collagenase(MMP1), 72kD gelatinase (MMP2) and stromelysin (MMP3) but not for 92kDgelatinase (MMP9) than did the control cells in the monolayer culture.Levels of mRNA for MMPs 1, 2 and 3 in the lattices were found to begreater than those in the monolayer cultures; over 100 times greater atcertain times during the contraction process (see Example 4 below).Gelatin zymography [Heussen & Dowdle 1980] was used to analyse andcompare production of the gelatinolytic components of the cells. It wasfound that gelatinolytic activity was increased compared to controls. Itappeared, therefore, that the increased mRNA production resulted inincreased protein production.

[0031] Antibodies against MMPs 1, 2, 3 and 9 were tested as contractioninhibitors. It was found that the antibodies against MMPs 1, 2 and 3gave significant inhibition of the contraction of collagen gels by humanocular fibroblasts but that the antibody against MMP9 did not result ininhibition of contraction.

[0032] In the present specification the term “contraction of collagen”includes not only shrinkage of collagen but also any remodelling ofcollagen that leads to contraction of the tissue comprising thatcollagen. It also includes contributions made by other components of thetissue, especially other extracellular matrix components, for example,elastin.

[0033] As indicated above, contraction of tissues comprisingextracellular matrix components, especially of collagen-comprisingtissues, may occur in connection with many different pathologicalconditions and with surgical or cosmetic procedures. Contracture maycause physical problems, which may lead to the need for medicaltreatment, or it may cause problems of a purely cosmetic nature. It istherefore very valuable to have medicaments capable of inhibiting, i.e.restricting, hindering or preventing, such contraction. Important usesof such medicaments are described below. It should be understood,however, that the uses according to the present invention are notrestricted to the manufacture of medicaments, or to methods oftreatment, medical or cosmetic, suitable for the conditions describedbelow. The present invention also includes use in the manufacture ofmedicaments or in methods of treatment suitable for use in any casewhere contraction of tissue comprising extracellular matrix componentsresulting substantially from extracellular matrix component contractionis occurring or may occur.

[0034] Although the discussion below refers specifically to collagencontraction and the use of collagenase inhibitors, broad spectrum MMPinhibitors and/or inhibitors of MMPs other than collagenases may be usedin inhibiting contraction of tissue comprising extra-cellular matrixcomponents and the present invention is to be understood as includingthe use of such MMP inhibitors as an alternative to the use ofcollagenase-specific inhibitors in the treatment of tissue comprisingextracellular matrix components. The present invention also includes theuse of broad spectrum MMP inhibitors and/or inhibitors of MMPs otherthan collagenases in addition to the use of collagenase inhibitors, forexample, in the treatment of tissue comprising extracellular componentsespecially collagen comprising-tissue.

[0035] Contraction of collagen-comprising tissue, which may alsocomprise other extracellular matrix components, frequently occurs in thehealing of burns. The burns may be chemical, thermal or radiation burnsand may be of the eye, the surface of the skin or the skin and theunderlying tissues. It may also be the case that there are burns oninternal tissues, for example, caused by radiation treatment.Contraction of burnt tissues is often a problem and may lead to physicaland/or cosmetic problems, for example, loss of movement and/ordisfigurement. The present invention therefore includes the use of MMPinhibitors, for example, collagenase inhibitors, for example, in theform of a medicament, to inhibit contraction of the burnt tissue as itheals.

[0036] A further aspect of the present invention is the inhibition ofthe contraction of skin grafts. Skin grafts may be applied for a varietyof reasons and may often undergo contraction after application. As withthe healing of burnt tissues the contraction may lead to both physicaland cosmetic problems. It is a particularly serious problem where manyskin grafts are needed as, for example in a serious burns case.

[0037] An associated area in which the medicaments and methods of thepresent invention are of great use is in the production of artificialskin. To make a true artificial skin it is necessary to have anepidermis made of epithelial cells (keratinocytes) and a dermis made ofcollagen populated with fibroblasts. It is important to have both typesof cells because they signal and stimulate each other using growthfactors. A major problem up until now has been that the collagencomponent of the artificial skin often contracts to less than one tenthof its original area when populated by fibroblasts. MMP inhibitors, forexample, collagenase inhibitors may be used to inhibit the contractionto such an extent that the artificial skin can be maintained at apractical size.

[0038] One area of particular interest is the use of MMP, e.g.collagenase,inhibitors to prevent or reduce contracture of scar tissueresulting from eye surgery. Glaucoma surgery to create new drainagechannels often fails due to scarring and contraction of tissues. Amethod of preventing contraction of scar tissue formed in the eye, suchas the application of a suitable agent, is therefore invaluable. Such anagent may also be used in the control of the contraction of scar tissueformed after corneal trauma or corneal surgery, for example laser orsurgical treatment for myopia or refractive error in which contractionof tissues may lead to inaccurate results. It is also useful in caseswhere scar tissue is formed on/in the vitreous humor or the retina, forexample, that which eventually causes blindness in some diabetics andthat which is formed after detachment surgery, called proliferativevitreoretinopathy. Other uses include where scar tissue is formed in theorbit or on eye and eyelid muscles after squint, orbital or eyelidsurgery, or thyroid eye disease and where scarring of the conjunctivaoccurs as may happen after glaucoma surgery or in cicatricial disease,inflammatory disease, for example, pemphigoid, or infective disease, forexample, trachoma. A further eye problem associated with the contractionof collagen-comprising tissues for which the methods and medicaments ofthe present invention may be used is the opacification and contractureof the lens capsule after cataract extraction.

[0039] Cicatricial contraction, contraction due to shrinkage of thefibrous tissue of a scar, is common. In some cases the scar may become avicious cicatrix, a scar in which the contraction causes seriousdeformity. A patient's stomach may be effectively separated into twoseparate chambers in an hour-glass contracture by the contraction ofscar tissue formed when a stomach ulcer heals. Obstruction of passagesand ducts, cicatricial stenosis, may occur due to the contraction ofscar tissue. Contraction of blood vessels may be due to primaryobstruction or surgical trauma, for example, after surgery orangioplasty. Stenosis of other hollow visci, for examples, ureters, mayalso occur. Problems may occur where any form of scarring takes place,whether resulting from accidental wounds or from surgery. Medicamentscomprising MMP inhibitors, e.g. collagenase inhibitors, may be usedwherever scar tissue is likely to be formed, is being formed or has beenformed.

[0040] Conditions of the skin and tendons which involve contraction ofcollagen-comprising tissues include post-trauma conditions resultingfrom surgery or accidents, for example, hand or foot tendon injuries,post-graft conditions and pathological conditions, such as scleroderma,Dupuytren's contracture and epidermolysis bullosa. Scarring andcontraction of tissues in the eye may occur in various conditions, forexample, the sequelae of retinal detachment or diabetic eye disease (asmentioned above). Contraction of the sockets found in the skull for theeyeballs and associated structures, including extra ocular muscles andeyelids, may occur if there is trauma or inflammatory damage. Thetissues contract within the sockets causing a variety of problemsincluding double vision and an unsightly appearance.

[0041] Although the above discussion relates in particular to humans,animals may exhibit the conditions described above or similar oranalogous conditions. The present invention therefore also relatesanalogously to medicaments and methods for use in veterinary practicefor the treatment and care of animals and especialy for use in thetreatment and care of mammals.

[0042] The present invention provides a method of treating a human orother mammal to inhibit contraction of tissue comprising anextracellular matrix component, especially contraction associated with achemical burn, a thermal burn or a radiation burn, a skin graft, apost-trauma condition resulting from surgery or an accident, glaucomasurgery, diabetes associated eye disease, scleroderma, Dupytren'scontracture, epidermolysis bullosa or a hand or foot tendon injury,which comprises administering to the human or other mammal atherapeutically effective amount of an MMP inhibitor.

[0043] It appears that MMP inhibitors, e.g. collagenase inhibitors,inhibit contraction tissues comprising extracellular components, forexample, collagen, caused by cells such as fibroblasts but do not appearto be able to bring about significant reversal of such contraction.Accordingly, tissue which is being affected should generally be treatedat the time when the contraction is occurring. Preferably treatmentshould take place as early as possible, advantageously as soon as, andmost advantageously before, the first signs of contraction are observed.In treatments, conditions or healing processes where contraction ofextracellular component, e.g. collagen, comprising tissue is common. MMPinhibitors, e.g. collagenase inhibitors, may be used as a routineprophylactic measure before any signs of contraction have actually beenseen.

[0044] Since active contraction appears to be associated with activeproduction of MMPs, the treatment used to prevent the contraction shouldbe continued over at least the period during which contraction is likelyto occur. This may often be quite a significant period of time, forexample, several years or even longer. Contraction may still occur evenafter an initially open wound appears to have healed, for example, inpatients with burns. Also conditions such as hand tendon contractioninvolve contraction even though there is no wound as such.

[0045] As indicated above, the present invention involves the use of MMPinhibitors, especially collagenase inhibitors.

[0046] Both natural and synthetic MMP inhibitors (inhibitors of enzymeactivity), including collagenase inhibitors, are known.Naturally-occurring MMP inhibitors include a₂-macrogl,obulin, which isthe major collagenase inhibitor found in human blood [Eisen et al 1970].Naturally occurring MMP inhibitors are also found in tissues. Thepresence of tissue inhibitors of MMPs has been observed in a variety ofexplants and in monolayer cultures of mammalian connective tissue cells[Vater et el 1979 and Stricklin and Wegus 1983]. Not only collagenaseinhibitors but also inhibitors for other MMPs, for example, gelatinaseand proteoglycanase are found. MMP inhibitors are generally unable tobind the inactive (zymogen) forms of the respective enzymes but complexreadily with active forms [Murphy et al 1981). Tissue MMP inhibitors arefound, for example, in dermal fibroblasts, human lung, gingival, tendonand corneal fibroblasts, human osteoblasts, uterine smooth muscle cells,alveolar macrophages, amniotic fluid, plasma, serum and the a-granule ofhuman platelets [Stricklin and Wegus 1983; Welgus et al 1985; Welgus andStricklin 1983; Bar-Sharvit et al 1985; Wooley et al; 1976; and Cooperet al 1985].

[0047] Synthetic collagenase inhibitors and inhibitors for other MMPshave been and are being developed. Compounds such as EDTA, cysteine,tetracycline and ascorbate are all inhibitors of collagenases but arerelatively non-specific. As indicated above, synthetic inhibitors thathave defined specificity for MMPs, including collagenase inhibitors, aredescribed in the literature. For example, U.S. Pat. Nos. 5,183,900,5,189,178 and 5,114,953 describe the synthesis ofN-[2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophanmethylamide, also known as GM6001 or Galardin (trade name), and otherMMP inhibitors. Other collagenase inhibitors based on hydroxamic acidare disclosed in WO 90/05716, WO 90/05719 and WO 92/13831. Furthersynthetic MMP inhibitors and in particular collagenase inhibitors thathave been developed include those described in EP-A-126,974 andEP-A-159,396 and in U.S. Pat. Nos. 4,599,361 and 4,743,587. Yet anotherinhibitor is BB-94, also known as Batimastat (British Bio-technologyLtd.), see for example, EP-A-276436. Disclosed in WO90/05719 as havingparticularly strong collagenase inhibiting properties are[4-(N-hydroxyamino)-2R-isobutyl-3S-(thio-phenyl-thiomethyl)succinyl]-L-phenylalanine-N-methylamide(especially good) and[4-(N-hydroxyamino)-2R-isobutyl-3S-(thiomethyl)succinyl]-L-phenylalanine-N-methylamideand in WO90/05716[4-(N-hydroxyamino)-2R-isobutylsuccinyl]-L-phenylalanine-N-(3-aminomethylpyridine)amide and[4-N-hydroxyamino)-2R-isobutyl-3S-methylsuccinyl]-L-phenylalanine-N-[4-(2-aminoethyl)-morpholino]amide.

[0048] The contents of the patent specifications and literaturereferences mentioned herein are hereby incorporated by reference.

[0049] As indicated above, the properties of natural and syntheticcollagen inhibitors may vary. Individual inhibitors often have differentspecificities and potencies. Some inhibitors are reversible, others areirreversible. In general the more potent an inhibitor's inhibitoryeffects on a collagenase the better. For some uses an inhibitor specificto one particular collagenase may be required but generally a broadspectrum MMP inhibitor, for example, GM6001 (Galardin (trade name)), ispreferred.

[0050] GM6001 (Galardin (trade name)) is a very potent MMP inhibitorthat is effective against collagenase. It has the structure:

[0051] A detailed account of its ability to inhibit human skinfibroblast collagenase, thermolysin and Pseudomonas aeruginosa elastaseis given in Grobelny et al, 1992. Its inhibition constants with threetypes of MMPs are now calculated to be: collagenase Ki = 0.4 nmol/1gelatinase Ki = 0.4 nmol/1 stromelysin Ki = 20 nmol/1

[0052] Preferred MMP inhibitors for use according to the presentinvention include GM6001 (Galardin) and those synthetic inhibitorsdescribed and referred to above. Preferred inhibitors include peptidehydroxamic acids or pharmaceutically acceptable derivatives thereof.Especially preferred are those compounds that are described and claimedin U.S. Pat. Nos. 5,189,178; No. 5,183,900 or No. 5,114,953 and that arecollagenase inhibitors. Those with low Ki values, i.e. high pKi valuesare generally preferred. GM6001 (Galardin (trade name)) is an MMPinhibitor that is especially preferred because it is one of the mostpotent collagenase inhibitors known at present. However, for certainapplications it may be preferable to use a less potent (weaker)inhibitor.

[0053] The preferred broad spectrum MMP inhibitor for use in accordancewith the present invention is GM6001 (Galardin (trade name)). It is ableto inhibit the action not only of collagenases but of other MMPs aswell.

[0054] Also preferred are inhibitors that are capable of inhibiting MMPs1, 2 and 3 (collagenase, 72kD gelatinase and stromelysin, respectively).These may be used individually or in combination.

[0055] As indicated above, an anti-MMP polyclonal or monoclonalantibody, especially an anti-collagenase antibody, may be used as aninhibitor. An MMP antigen may be used in immunisation protocols toobtain polyclonal antisera immunospecific for that enzyme. The antigenmay be a hapten derived from an MMP, especially from an active siteregion, or may be a full-length MMP or a fragment thereof. Usingstandard protocols and mammalian subjects, such as rabbits or mice,polyclonal antibodies may be obtained. Those may then be used asinhibitors. Monoclonal antibodies may be produced according to standardprocedures, for example, using an appropriate MMP antigen, for example,a collagenase antigen.

[0056] Antibodies which are specific for a particular MMP may be madeand the use of such specific inhibitors may be preferred under certaincircumstances. For example, an antibody to MMP1, MMP2 or MMP3(collagenase, 72kD gelatinase or stromelysin respectively) or a mixtureof two or more thereof may be used.

[0057] An alternative method of inhibiting the action of an MMP is toreduce the amount of the MMP by preventing its production. One method ofpreventing protein production is by the use of antisense nucleic acidmolecules. An antisense molecule need not be large; 20 base pairs isoften sufficient. If the molecule is small it should be able to enterthe cells unaided but liposomes can be used to assist entry if required.An antisense molecule will usually be designed to attach to the MMP mRNAbut may be designed to attach to the appropriate DNA during replicationand transcription.

[0058] Although antibodies usually bind to proteins it is possible toproduce antibodies which bind to nucleic acids. Accordingly, there maybe used as an inhibitor according to the present invention an antibodythat binds to the mRNA or the DNA of the selected MMP and hence hindersproduction of the MMP.

[0059] Inhibition by reducing production of an MMP has the advantagethat it should be possible to use a smaller amount of inhibitor than isrequired for direct inhibitors of MMP enzyme activity because each MMPmRNA molecule and the MMP DNA is responsible for the production of manyMMP enzyme molecules.

[0060] Inhibitors for use according to the present invention must beable to be used in high enough concentrations and large enough doses togive adequate inhibition without being toxic to cells with which theycome into contact.

[0061] For the treatment of some conditions it may be preferred to use amedicament containing a collagenase inhibitor and at least one otherenzyme inhibitor. That additional inhibitor may also have collagenaseinhibitory properties and/or it may have inhibitory properties for adifferent enzyme, for example, for a different MMP. If two or moreinhibitors are used then the second and any additional inhibitorpreferably has inhibitory properties for an enzyme other than acollagenase. Additional inhibitors may be, for example, inhibitors ofother MMPs such as a gelatinase or a stromelysin, or inhibitors of otherenzymes that break down tissue such as serine proteases, for example, aserine protease inhibitor such as aprotinin may be used.

[0062] Cytokines, for example, interleukin-l, in the environment ofcollagen-comprising tissue may stimulate collagenase production and soit may also be preferable to include an inhibitor of cytokines in themanufacture of medicaments or in methods of treatment according to thepresent invention.

[0063] Medicaments according to the present invention are generallyprovided in a pharmaceutical preparation form suitable for topicaladministration, for example, an emulsion, suspension, cream, lotion,ointment, drops, foam or gel. Such preparations are generallyconventional formulations, for example, as described in standard textbooks of pharmaceutics such as the British Pharmacopoeia. Other suitablepharmaceutical forms for topical administration include dry powders,aerosols and sprays, which may be especially suitable for application toburns. Further suitable pharmaceutical preparation forms include thosefor administration by injection or infusion, for example, sterileparenteral solutions or suspensions, especially for administrationdirectly into, or into the area of, the extracellular matrix component,e.g. collagen, comprising tissue, for example, by subconjunctival,subcutaneous, interpleural or intra-peritoneal injection, and also slowrelease delivery systems, for example, liposome systems.

[0064] The invention especially provides a pharmaceutical preparation,other than a preparation suitable for use in the eye, suitable forapplication to a wound (including an ulcer, a burn or skin graftcomprising a matrix metalloproteinase inhibitor, advantageously thepreparation is in the form of a solution, suspension, cream, ointment,or gel, in which the MMP inhibitor is in a concentration of from 0.4μg/ml to 400 μg/ml. The MMP inhibitor is preferably a collagenaseinhibitor.

[0065] Oral formulations may also be used. These may be in the form oftablets, capsules, powders, granules, lozenges or liquid or gelpeparations. Tablets may be coated by methods well known in normalpharmaceutical practice. Liquid formulations include syrups. Oralformulations may be used to treat directly conditions such as stomachulcers and may also be used to treat conditions systemically.

[0066] The inhibitor(s) may be dissolved or dispersed in a diluent orcarrier. The choice of carrier depends on the nature of the inhibitor,its solubility and other physical properties, and on the method and siteof application. For example, only certain carriers are suitable forpreparations for use in the eye.

[0067] Carriers include ethylene glycol, silver sulphadiazine cream andhypromellose. These may be used in creams and drops. An acetate buffersystem may also be used. Further pharmaceutically suitable materialsthat may be incorporated in pharmaceutical preparations includeabsorption enhancers, pH regulators and buffers, osmolarity adjusters,emollients, dispersing agents, wetting agents, surfactants, thickeners,opacifiers, preservatives, stabilizers and antioxidants, foaming agentsand flocculants, lubricants, colourants and fragrances (generally onlyin primarily cosmetic preparations).

[0068] Gels and liposomes may be the preferred delivery method when theinhibitor is an antisense molecule.

[0069] Preferably a medicament according to the present invention isapplied directly to an open wound or is injected directly into the siteof tissue contraction. Suitable medicaments may, however, be applied tothe skin surface where the tissue to be treated is below that surface,the active ingredient then being absorbed by and passing through theskin. Penetration enhancers are preferably incorporated in suchmedicaments.

[0070] Medicaments according to the present invention comprising MMPinhibitors, for example, collagenase inhibitors, for use in theinhibition of the contraction of tissues comprising extracellular matrixcomponents, for example, collagen-comprising tissues, may containfurther pharmaceutically active ingredients, for example antibiotics,antifungals, steroids, and further enzyme inhibitors, for example,serine protease inhibitors (as described above). Further components forcertain indications include growth or healing promoters such asepidermal growth factor (EGF), fibronectin and aprotinin. As mentionedabove cytokine inhibitors may also be included.

[0071] The inhibitors will generally be used in liquid and othernon-solid formulations having concentrations of around 0.4 to 400 μg/ml.In some cases, however, higher concentrations may be required. The totalamount used and the dose administered will depend on the severity andarea of the contraction, the condition causing it and the physicalcharacteristics of the patient and the site and method ofadministration.

[0072] The following non-limiting Examples illustrate the invention. TheExamples relate to experiments carried out in vitro; the teachings aredirectly applicable in vivo, for example, to the treatment of humans andmammals, for example, as described in the specification.

[0073] The Examples illustrate the use of MMP inhibitors in in vitromodels of scar contraction and of artificial skin. They also includeexperiments which illustrate the lack of toxicity to cells of MMPinhibitors and their effect on cell morphology. Further experimentsinvestigate the levels of some MMP mRNA and of some MMPs present incells during contraction.

[0074]FIG. 1 is a diagrammatic representation of the results of theexperiments described in Example 1. It is a graph showing the area offibroblast populated collagen gel over time for a number of differentculture regimes.

[0075]FIG. 2 is a diagrammatic representation of the results of theexperiments described in Example 2. It is a histogram showing counts perminute of radiation emitted by cells grown under a number of differentconditions.

[0076]FIG. 3A is a diagrammatic representation of the results ofexperiments described in Example 3(a). It is a graph showing meanlattice area of fibroblast populated collagen gels over time fordifferent culture regimes.

[0077]FIG. 3B is a diagrammatic representation of the results ofexperiments described in Example 3(a). It is a graph showing meanlattice area of fibroblast populated collagen gels over time fordifferent culture regimes.

[0078]FIG. 3C is a diagrammatic representation of the results ofexperiments described in Example 3(b). It is a-graph showing meanlattice area of fibroblast populated collagen gels over time fordifferent culture regimes.

[0079]FIG. 3D is a diagrammatic representation of the results ofexperiments described in Example 3(c). It is a graph showing meanlattice area of fibroblast populated collagen gels over time fordifferent culture regimes.

[0080]FIG. 3E is a diagrammatic representation of the results ofexperiments described in Example 3(c). It is a graph showing the meannumber of cells per well against time under a number of differentculture regimes.

[0081]FIG. 3F is a diagrammatic representation of the results ofexperiments described in Example 3(c). It is a graph showing thepercentage of viable cells against time under a number of differentculture regimes.

[0082]FIG. 3G is a diagrammatic representation of the results ofexperiments described in Example 3(d). It is a graph showing meanlattice area of fibroblast populated collagen gels over time fordifferent culture regimes.

[0083]FIG. 3H is a diagrammatic representation of the results ofexperiments described in Example 3(c). It is a graph showing meanlattice area of fibroblast populated collagen gels over time fordifferent culture regimes.

[0084]FIG. 4A is a diagrammatic representation of the results of theexperiments described in Example 5(i). It is a graph showing the meanlattice area of fibroblast populated collagen gels over time under twodifferent culture regimes.

[0085]FIG. 4B is a diagrammatic representation of the results of theexperiments described in Example 5(ii). It is a graph showing the meanlattice area of fibroblast populated collagen gels over time under twodifferent culture regimes.

[0086]FIG. 4C is a diagrammatic representation of the results of theexperiments described in Example 5(iii). It is a graph showing the meanlattice area of fibroblast populated collagen gels over time under twodifferent culture regimes.

[0087]FIG. 5 is a diagrammatic representation of the results of theexperiments described in Example 6. It is a graph showing the meanlattice area of fibroblast populated collagen gels over time under twodifferent culture regimes.

[0088]FIG. 6 is a reproduction of a photograph showing the results ofthe PCR reactions described in Example 4.

[0089]FIG. 7 is a reproduction of a photograph showing the results-ofthe gelatin zymography analyses described in Example 4.

[0090]FIG. 8A and FIG. 8B are each a reproduction of a photograph of acollagen gel seeded with fibroblasts showing the degree of contractionas described in Example 8.

[0091]FIG. 8A shows a control gel and

[0092]FIG. 8B shows a gel exposed to inhibitor.

[0093]FIG. 9A and FIG. 9B are each a reproduction of a photograph of acollagen gel seeded with fibroblasts showing the development of actinstress fibres on the surface of the cells as described in Example 8.

[0094]FIG. 9A shows a control gel and

[0095]FIG. 9B shows a gel exposed to inhibitor.

[0096]FIG. 10A and FIG. 10B are each a reproduction of a photograph offibroblasts grown in monolayers showing the presence of stress fibres asdescribed in Example 8.

[0097]FIG. 10A shows a control gel and

[0098]FIG. 10B shows a monolayer exposed to inhibitor.

[0099]FIG. 11A and FIG. 11B are each a reproduction of a photograph of acollagen gel seeded with fibroblasts showing cellular attachments asdescribed in Example 8.

[0100]FIG. 11A shows a control gel and

[0101]FIG. 11B shows a gel exposed to inhibitor.

EXAMPLES Example 1

[0102] Preparation of Materials

[0103] (a) Collagen Solutions

[0104] Solutions of type 1 collagen were prepared by dissolving 100 mgof collagen in 20 ml of 0.1% (v/v) glacial acetic acid in distilledwater. The collagen was sigma type 1 collagen and was prepared by themethod of Bornstein MB Lab Invest 7134 1958.

[0105] (b) Concentrated Culture Medium

[0106] A concentrated tissue culture medium was prepared by mixing thefollowing:

[0107] 35 ml distilled water

[0108] 15 ml 10× MEM (Eagle's minimal essential medium)

[0109] 15 ml glutamine

[0110] 1.5 ml fungizone (amphotericin B 250 μg/ml of water)

[0111] 1.5 ml 10,000 units penicillin/10 mg streptomycin per ml solution(solvent is water)

[0112] 4 ml 7.5% (w/v) sodium bicarbonate solution

[0113] Solutions of 4.9 ml of the concentrated culture medium in 180 μlof 0.1M sodium hydroxide solution were used in the preparation ofcollagen gels.

[0114] (c) Collagenase Inhibitor Solutions and Control Solutions

[0115] Solutions having three different concentrations of collagenaseinhibitor (400, 40 and 4 μg/ml), a buffer solution (control 1) and anormal growth medium solution (control 2) were prepared as describedbelow. The collagenase inhibitor used was GM6001 (Galardin (tradename)).

[0116] 50 μl of glacial acetic acid was added to 11 mg of collagenaseinhibitor and the inhibitor was allowed to dissolve. The pH of a 24.875ml aliquot of serum-free HEPES (N-[2-hydroxyethylpiperazine-N′-[2-ethanesulfonic acid]) buffered DMEM (Dulbecco's Modified Eagle's Medium) wasadjusted to pH 8 using sterile 1M sodium hydroxide solution and thealiquot was then added to the inhibitor solution. The resulting solutionhad an inhibitor concentration of 400 μg/ml.

[0117] The 400 μg/ml solution was serially diluted with serum-freeHEPES-DMEM to give solutions containing the collagenase inhibitor atconcentrations of 40 and 4 μg/ml.

[0118] The solutions of all three concentrations were then supplementedwith 10% (v/v) newborn calf serum.

[0119] The buffer solution (control 1) was prepared by adding 50 μl ofglacial acetic acid to a 24.875 ml aliquot of serum-free HEPES bufferedDMEM of pH 8 (pH adjusted with sterile 1M sodium hydroxide solution asabove), then readjusting the pH to pH 7.4 with 1M sterile sodiumhydroxide solution and finally supplementing with 10% (v/v) of newborncalf serum (NCS).

[0120] The normal growth medium solution (control 2) was prepared bysupplementing an aliquot of serum-free HEPES buffered DMEM with 10%(v/v) newborn calf serum.

[0121] The pH and osmolarity of the test and control solutions weremeasured, see Table 1, Treatment of Gels with Test Solutions.

[0122] (d) Cell Cultures

[0123] Cultures of human ocular fibroblasts were grown, in the normalway (see Khaw P. T. et al 1992 for the method of growth), until themonolayers (single layers of cells on a plastic culture disc, notembedded in a matrix) were just subconfluent. They were then removedfrom their substratum via a trypsinisation and were pelleted bycentrifugation at 3000× g for 8 minutes. The supernatant was thendiscarded and the cell pellet resuspended in 1.1 ml of newborn calfserum. 100 μl of the suspension was removed and counted in a Coultercounter (Model ZF).

[0124] (e) Collagen Gels

[0125] Each collagen gel had a final volume of 1.1 ml made up of 0.6 mlof a solution of type 1 collagen obtained according to (a) above, 0.35ml of concentrated culture medium prepared according to (b) above, and0.15 ml of cell suspension containing 100,000 cells prepared asdescribed in (d) above.

[0126] Triplicate gels were made by adding 1.05 ml of the concentratedmedium to 1.8 ml of the type 1 collagen solution, mixing rapidly andthen adding 0.45 ml of a cell suspension containing 300,000 cells andmixing rapidly.

[0127] 1 ml aliquots of this gel were then added to petri dishes (area=8cm²) and then the dishes were rotated so that the gels were evenlydistributed across the bottoms of the dishes. The gels were thenincubated at 37° C. in 5% CO₂ in air until they had solidified (usually3 to 5 minutes). An area of the gel was then detached from the edge ofeach petri dish and 3 ml of normal growth medium solution (control 2),inhibitor test solution or buffer solution (control 1), were added. Eachgel was then fully detached from the edges and bottom of the petri dishso that free-floating gels were obtained.

[0128] Treatment of Gels with Test Solutions

[0129] The gels, after solidification, were treated for 24 hours with 3ml of each of the following test solutions: TABLE 1 Test Solution pHOsmolarity 1) Collagenase inhibitor at 400 μg/ml 7.8 421 2) Collagenaseinhibitor at 40 μg/ml 7.8 339 3) Collagenase inhibitor at 4 μg/ml 7.9329 4) Buffer (control 1) 7.8 404 5) Normal growth medium (control 2)7.8 331

[0130] The test and control solutions were replaced by 3 ml of theappropriate fresh solution after 24 hours. Photographs of the gel areaswere taken on days 1 and 2 and were then digitised. The resulting datawere processed by a computer and the gel areas were calculated.Phase-contrast photographs were taken of the top and middle of each gelafter 1 and 2 days.

[0131] Results

[0132] The changes in area observed for the collagen gels over the 2 daytest period are shown in FIG. 1. (In FIG. 1 mcg represents micrograms.)It can be seen from the results that all the solutions of collagenaseinhibitor inhibited the contraction of the collagen gels in comparisonwith the buffer solution (control 1) and the normal growth mediumsolution (control 2). The amount of inhibition observed was dosedependent; the solution with the highest inhibitor concentration showedthe strongest inhibition of gel contraction. The buffer solution(control 1) also showed some inhibitory properties in comparison withthe normal growth medium solution (control 2) but the effect was notsignificant when compared to that exhibited by the buffered collagenaseinhibitor solution of even the lowest concentration. At the end of thetests the cells in the collagen gels were still alive and phase contrastmicroscopy showed that they appeared to be viable.

[0133] Discussion

[0134] It appears that MMPs are involved in fibroblast mediatedcontraction of collagen and that the use of an inhibitor can restrictcontraction without killing the cells. Furthermore, the amount ofinhibition is dose dependent.

Example 2

[0135] The toxicity of GM6001 (Galardin (trade name)) and a secondcollagenase inhibitor GM1489 (a derivative of GM6001) to fibroblasts wastested at various concentrations.

[0136] The toxicity of the inhibitors was tested by measuring DNAsynthesis by tritiated thymidine incorporation. The assay was adaptedfrom a procedure previously described, Woost, P. G. et al 1992.

[0137] Human ocular fibroblasts (Tendon's capsule fibroblasts) werecultured in Trimix (DMEM/F-10/M-199) cell culture medium (available fromGIBCO/BRL) supplemented with 10% bovine calf serum and grown toconfluency in T-75 tissue culture flasks. (That is flasks having asurface area of 75 cm².) The cells were split and seeded into 24 wellplates at a density of 1×10⁴/well in 1.0 ml of medium with 10% bovinecalf serum. The cells were incubated for 24-36 hours or until theyreached 60-70% confluency. The medium was then changed to serum-freeTrimix and the cells were incubated for an additional 24 hours. Thecells were then incubated in quadruplicate wells for 24 hours with 1.0μCi/well ³H-thymidine in the following conditions: serum-free medium(Trimix), medium plus 10% serum, medium plus 10% serum and vehicle,alone or with the inhibitors. The vehicle was a buffered acetatesolution. The solutions comprising medium with 10% serum and vehicle,alone or with inhibitors, were prepared by the method described inExample 1, section (c), above, using Trimix instead of the HEPESbuffered DMEM. The inhibitors, GM6001 and GM1489, were each tested atconcentrations of 80.0, 8.0, and 0.8 μg/ml. At the end of the incubationperiod, the wells were washed 3 times with PBS and fixed with 12.5% TCAfor 10 min followed by methanol for 10 min. The plates were air driedand the cells solubilized in 1.0ml of 0.2 N NaOH at 37° C. for 1 hour.Radioactivity was determined by liquid scintillation counting 900 ml ofthe solubilized cells. The experiments show that there is no significanttoxicity at any of the concentrations tested. The results are shown inFIG. 2. In FIG. 2 SF1 and SF2 stand for serum-free medium, 10% formedium plus 10% serum, V1 and V2 for medium plus 10% serum and vehicleand the other results are for the inhibitor solutions (comprising alsomedia plus 10% serum and vehicle).

[0138]FIG. 2 shows the thymidine incorporation of the ocular fibroblastsat different concentrations of each collagenase inhibitor.

[0139] Discussion

[0140] It can be seen that there is no significant reduction inthymidine uptake even with the highest concentrations of the inhibitors.This indicates that the reduction in collagen contraction found whenusing collagenase inhibitors is not due to inhibition of cellproliferation.

[0141] In the following Examples methods and materials substantially asdescribed in Example 1 are used except where it is indicated to thecontrary.

Example 3

[0142] This experiment was designed to investigate the effect of thefollowing MMP inhibitors: Galardin, BB-94 and antibodies to MMPs, 1, 2 3and 9, on ocular fibroblast mediated collagen contraction.

[0143] The preparation of materials was as described in Example 1 aboveexcept where indicated. Test solutions were used in approximately 3 mldoses. For all contraction experiments cell morphology was monitored byphase contrast microscopy and the growth medium was changed every 3 to 4days.

[0144] a) Cell Number Dependence

[0145] Collagen gels were seeded with either 100,000 or 500,000 cells.They were exposed to inhibitor and control solutions comprising 40 μg/mland 4 μg/ml of Galardin or equimolar hydroxamic acid (control) at anequivalent to 40 μg/ml of Galardin (100 μM) (made up as described inExample 1(c) above). The gels were monitored for 7 days.

[0146] For results, see FIGS. 3A and 3B.

[0147]FIG. 3A shows the results for the collagen lattices (gels)populated with 500,000 cells per lattice. The mean lattice area isplotted against time (in days) for gels treated with each of the threesolutions. As may be seen, the contraction of the gels exposed toinhibitor solutions is much less than that of gels exposed to thecontrol solution. FIG. 3B shows the results for the collagen latticespopulated with 100,000 cells per lattice. Again the mean lattice area isplotted against time for each of the three regimes. As may be seen, thecontraction of the gels exposed to inhibitor solutions is much less thanthat of gels exposed to control solutions. Comparing FIGS. 3A and 3Bshows that the contraction of lattices populated with 500,000 cells perlattices is greater than that of lattices populated with 100,000 cellsunder the same conditions.

[0148] Discussion

[0149] The experiments confirmed that contraction is stronger when thereis a higher cell concentration and that the same dose of inhibitorcannot then provide such good inhibition as when there is a lower cellconcentration.

[0150] b) Reversibility

[0151] Collagen gels were prepared and seeded with fibroblasts (1×10⁵cells/lattice (gel)) as described in Example 1 above. They were exposedto inhibitor and control solutions in the following ways:

[0152] (i) continual exposure to control solution (medium containinghydroxamic acid);

[0153] (ii) continual exposure to inhibitor solution (medium containing4 μg/ml Galardin);

[0154] (iii) continual exposure to inhibitor solution (as (b)) until day14 post seeding and then replacement by control solution (as (a));

[0155] (iv) lattices were allowed to contract for 5 days and then werecontinually exposed to control solution (as (a)) for 25 days;

[0156] (v) lattices were allowed to contract for 5 days and then werecontinually exposed to inhibitor solution (as (b)) for 25 days;

[0157] (vi) lattices were allowed to contract for 5 days and then werecontinually exposed to inhibitor solution (medium containing 40 μg/mlGalardin) for 25 days.

[0158] The test solutions were made up in accordance with Example 1 (c)and the growth medium in the cultures was changed every 3 to 4 days.

[0159] The results of (i), (ii) and (iii) are shown in FIG. 3C and theresults of (iv), (v) and (vi) are shown in FIG. 3D.

[0160]FIG. 3C is a plot of mean lattice area against time showing theresults for experiments (i), (ii) and (iii). As may be seen, the meanarea of lattices treated with control solution decreases from day 0;contraction occurs for the whole period. For the lattices initiallytreated with inhibitor solutions there is very little contraction and inthe case where the lattices continue to be exposed to inhibitor (ii) thereduction in mean area is low even after 40 to 50 days. For the latticesexposed to inhibitor solution until day 14 (shown as * in FIG. 3C) andthereafter exposed to control solution (experiment (iii)) there is asignificant increase in contraction from day 14 onwards.

[0161]FIG. 3D is a plot of mean lattice area against time for each ofexperiments (iv), (v) and (vi). As may be seen, in the first 5 days ofthe experiments when lattices were not exposed to test solutions thelattices underwent substantial contraction. After day 5 (marked as * inFIG. 3D) the lattices were exposed to the test solutions. Thecontraction continued (there was no reversal) but at a reduced rate.Both the 4 μg/ml and 40 μg/ml solutions of Galardin inhibitedcontraction in comparison with the control solution. The 40 μg/mlsolution of Galardin gave a stronger inhibitory effect than the 4 μg/mlsolution.

[0162] Discussion

[0163] Experiments (i), (ii) and (iii) showed that exposure to inhibitorsolution inhibited contraction in comparison with exposure to thecontrol solution and that when the inhibitor solution was then replacedby control solution the rate of contraction increased, i.e. inhibitionstopped. Hence the inhibition is reversible.

[0164] Experiments (iv), (v) and (vi) showed that even when contractionof the gels had been allowed to take place for 5 days without any typeof inhibition, the addition of inhibitor solutions still gave inhibitionof contraction. The more concentrated inhibitor solution gave greaterinhibition.

[0165] c) Cytotoxicity

[0166] This experiment was carried out substantially according to themethod of Example 2, except that cells were seeded at 2×10⁴ cells perwell, cultured without radioactive material and were exposed to controlmedium (DMEM/10% NCS) or Galardin at 40 μg/ml and 4 μg/ml concentration.Approximately 1 ml of test solution was added in each case. Wells wereharvested and cells counted on a haemocytometer, see FIG. 3E for theresults, and the percentage of viable cells in each well was measuredusing the trypan blue dye exclusion test, see FIG. 3F for the results.As can be seen the Galardin did not adversely affect cell viability andnumber compared to controls.

[0167] Discussion

[0168] It appears that MMP inhibitors may be used in quantities andconcentrations high enough to restrict contraction without significanttoxicity.

[0169] d) BB-94

[0170] The experiment was carried out as for (a) above with 1×10⁵ cellsper lattice and the test solutions used were BB-94 4 μg/ml, BB-94 0.4μg/ml and control with vehicle (0.1% vol/vol DMSO). The development ofthe lattices was followed for seven days after seeding. For the resultssee FIG. 3G, from which it is clear that BB-94 gave significantinhibition of contraction.

[0171] e) Antibodies

[0172] The experiment was carried out as for (d) above except that thetest solutions used were 1:50 (vol/vol) of antibodies in growth medium.The antibodies used were antibodies to MMPs 1, 2, 3 and 9, obtained fromBiogenesis Ltd., (Poole, U.K.). A control solution of 0.1% sodium azideand 0.14% rabbit serum in PBS diluted to 1:50 in growth medium was alsoused. See FIG. 3H for the results.

[0173]FIG. 3H is a plot of mean lattice area against time for latticesexposed to each of the test solutions. As may be seen, the latticesexposed to control solution and to the solution of antibody to MMP9showed significant contraction. The lattices exposed to one of theantibodies to MMP1, MMP2 or MMP3 showed no significant contraction.

[0174] Discussion

[0175] As may be seen from FIG. 3H significant inhibition was achieved.with antibodies for MMPs 1, 2 and 3 but not with the antibody for MMP9.This therefore shows that antibodies may act as satisfactory MMPinhibitors for use in inhibiting contraction. It also appears that whileMMPs 1, 2 and 3 may all be involved in the contractile process MMP9 isnot.

Example 4

[0176] A series of experiments were carried out in which the MMP mRNAand protein production of human Tenon's capsule fibroblasts, under anumber of conditions, was analysed.

[0177] Fibroblast cells were both grown in monolayer culture and wereseeded in collagen lattices. Total RNA was isolated from samplescontaining 3×10⁶ cells using the method described in Chomczynski &Sacchi 1987. Samples were taken from cells in monolayer culture on day 0of the experiment and samples were also taken from cells, which werecontracting collagen lattices after 9 hours, 1 day and 7 days.

[0178] 1 μg/mg samples of the RNA were treated with known copy numbersof a synthetic RNA template (0.1 to 100,000 copies) containingcomplementary sequences to the primers for sequences to MMPs 1, 2, 3 and9. The mixtures underwent reverse transcriptase reactions prior toamplification by PCR using specific primers for MMPs 1, 2, 3 and 9.During the PCR reaction both the sample RNA and the synthetic templateRNA compete equally for primer binding.

[0179] Band intensities were image analysed and the ratio of amplifiedsynthetic template to amplified sample intensities calculated. Thesevalues were plotted versus initial template copy number. When the ratioof amplified synthetic template to sample is one, then the initial copynumbers of synthetic template and sample are equal. This allowedcalculation of message copy number per cell in all samples.

[0180] Samples of conditioned medium and collagen lattices (1×10⁵cells/lattice) were collected on days 1, 3 and 7 post seeding. Latticeswere washed in phosphate buffered saline (PBS; 3×3 ml) prior tohomogenisation in 0.5% (vol/vol) Triton-XlOo in PBS (Hunt et al. 1993].Gelatin zymography [Heussen & Dowdle 1980] was then performed on thesesamples. Briefly, samples and prestained molecular weight standards wereresolved on 10% tris glycine gels containing 0.1% gelatin (Novex, R&DSystems, Oxford, U.K.). Gels were then incubated for 30 minutes inrenaturing buffer, 30 minutes in developing buffer followed by a further18 hours in developing buffer at 37° C. (all Novex). Gels were thanstained in 0.5% (wt/vol) commassie blue in 45% (vol/vol) water, 45%(vol/vol) methanol and 10% (vol/vol) glacial acetic acid, followed bydestaining in 45% (vol/vol) water, 45% (vol/vol) water, 45% (vol/vol)methanol and 10% (vol/vol) glacial acetic acid. MMP activity appeared asclear bands on a blue background.

[0181] Results

[0182] The experiments showed that the RNA extracted from cells whichwere contracting collagen contained raised levels of mRNA for MMPs 1, 2and 3 but not 9, in comparison with that taken from cells in monolayercultures. They also showed that levels of MMP mRNA decreased over the 7day culture period. mRNA Copy Number/10⁶ Cells MMP Monolayer 9 Hours 1Day 7 Days 1 8 71 143 3 2 <1 15 144 81 3 <1 18 132 16 9 <1 <1 <1 <1

[0183] Also see FIG. 6 for results. FIG. 6 is a reproduction of aphotograph of the results of the PCR reaction. It shows the sample andtemplate bands at day 0, 9 hours, 1 day and 7 days for mRNA encodingeach of MMPs1, 2, 3 and 9.

[0184] The gelatin zymography showed that actively contracting cellswhen compared to cells from monolayer cultures produced fourproteolytically active species (Mr approximately 100,000; 90,200; 72,000and 57,000), two of which appear (ones with Mrs of 72,000 and 57,000) toincrease over the 7 day period. See FIG. 7 for results. One wasactivated upon incubation with aminophenyl mecuric acetate (Mr 100,000reduced to 57,000).

[0185] Discussion

[0186] The experiments showed that both MMP mRNA and protein productionby ocular fibroblasts is dramatically increased upon culture within, andduring the contraction of, a three dimensional collagen matrix. Ittherefore appears that active production of MMPs occurs during thecontractile process.

[0187] Further experiments showed that treatment of seeded lattices withGalardin resulted in the abolishment of the four proteolytically activespecies.

Example 5

[0188] This series of experiments investigated the effect of MMPinhibitors on the degree of contraction of collagen lattices byfibroblasts from various tissue sites and species.

[0189] Collagen lattices (gels) were prepared as previously described inExample 1 and were seeded with 1×10⁵ cells per lattice of:

[0190] i) human dermal fibroblasts;

[0191] ii) rat parietal sheath fibroblasts; or

[0192] iii) rat endotendon fibroblasts.

[0193] The lattices were then exposed to growth medium containingGalardin at 40 μg/ml or hydroxamic acid at 100 μM (control). The testsolutions were made up as described in Example 3 above and approximately3 ml dose of the appropriate test solutions were used. The area of eachlattice was measured over 7 days and the results are shown in FIGS. 4A,4B and 4C.

[0194]FIGS. 4A, 4B and 4C are plots of mean lattice area over time forlattices populated with fibroblasts under exposure to either controlsolution or inhibitor solution. FIG. 4A for human dermal fibroblasts, 4Bfor rat parietal sheath fibroblast and 4C for rat endotendon fibroblast.As may be seen, the contraction of lattices populated with any of thethree types of fibroblasts was greatly reduced when exposed to theinhibitor solutions in comparison with the lattices populated with thesame type of fibroblasts and exposed to the control solution.

[0195] Discussion

[0196] Exposure to the inhibitor resulted in a significant degree ofinhibition of contraction compared to controls in all of the cell types.In each case, inhibition of contraction was accompanied by a decreasedcellular invasion into and migration through, the surrounding matrixcompared to controls. Therefore it appears that inhibition of cellularinvasion is an important mechanism of MMP inhibitor action.

Example 6

[0197] The effect of an MMP inhibitor on the contraction of anartificial skin equivalent was tested. A collagen lattice (gel) wasprepared as in Example 1 and was seeded with both human keratinocytesand human dermal fibroblasts to mimic skin. This lattice was thenexposed to growth medium containing Galardin at 40 μg/ml or hydroxamicacid at 100 μM (control). The solutions were made up as described inExample 3(a) above and approximately 3 ml portions of the appropriatesolutions were used. The lattice area was measured over 7 days and theresults are-shown in FIG. 5.

[0198]FIG. 5 is a plot of the mean lattice area over time (in days) forlattices exposed to the control solution and for lattices exposed to theinhibitor solution. As may be seen, the lattices exposed to theinhibitor solution suffered less contraction than those exposed to thecontrol solution.

[0199] Discussion

[0200] It is therefore possible to inhibit the contraction of a collagengel with fibroblasts and epithelial cells, i.e. an approximate model ofskin.

Example 7

[0201] The effects of MMP inhibitors on ocular fibroblast morphologywithin collagen lattices were studied in this series of experiments.

[0202] Collagen lattices seeded with ocular fibroblasts were preparedand exposed to MMP inhibitors, both synthetic chemicals and antibodies,as described in Example 3 above. Cellular morphology was monitored byphase contrast microscopy.

[0203] Cells populating control collagen lattices exhibited stellate (S)and bipolar (B) morphology by 7 days post seeding. Small cytoplasmicprojections were exhibited by cells exposed to 4 μg/ml Galardin, 4 μg/mlBB-94, antibody to MMP1 or MMP2 at 7 days post seeding. Cells exposed toMMP3 antibody did not produce any cytoplasmic projections into thesurrounding matrix. Exposure to antibody to MMP9 did not affectmorphology compared to controls.

[0204] Discussion

[0205] These results showed that cells populating lattices exposed toGalardin, BB-94 and antibodies to MMPs 1, 2 and 3 exhibited decreasedinvasion into the surrounding collagen matrix compared to controls andto cells exposed to an antibody to MMP9. This decrease in invasion intothe matrix was always accompanied by the inhibition of both latticecontraction and migration through the matrix. This again showed thatinhibition of invasion is important.

Example 8

[0206] This series of experiments was designed to -investigate theeffects of an MMP inhibitor (Galardin) on Tenons capsule fibroblastcellular processes required for collagen contraction.

[0207] Monolayer cultures of fibroblasts and collagen lattices (gels)seeded with 1×10⁵ cells per lattice were prepared (as described inExample 1).

[0208] The degree of contraction of collagen gels treated with controlsolution and with inhibitor solution containing 40 μg/ml Galardin(solutions were made up as described in Example 1 and approximately 3 mlportions of solutions were used) was monitored, as described above. FIG.8A shows the degree of lattice contraction of a control gel 7 days postseeding and FIG. 8B shows the degree of contraction of the gel which wasexposed to inhibitor solution.

[0209] The actin cytoskeleton of cells in lattices and in monolayerculture was immunofluorescently stained with FITC-phalloidin (Martin &Lewis 1992]. See FIGS. 9A (control) and 9B (exposed to inhibitor). Thearrows indicate the actin stress fibres on the surface of the cellspopulating the collagen gels. These fibres are clearly present in thecontrol but do not appear to be present in the lattice that was exposedto inhibitor solution.

[0210] Differences between the controls and those exposed to inhibitor(40 μg/ml Galardin) were seen. In order to assess whether thisdifference was owing to the direct effect of the inhibitor or todifferences in the degree of invasion into the matrix, cells wereexposed in monolayer culture to the inhibitor and the cytoskeletonwas-stained. See FIGS. 10A (control) and 10B (exposed to inhibitor). Thearrows indicate the stress fibres on the cells in the monolayer culture.As may be seen, they are present in both the control monolayer cultureand in the monolayer culture exposed to the inhibitor solution.

[0211] The inhibitor did not appear to affect the actin cytoskeletoncompared to controls in monolayer culture.

[0212] Lattices for transmission electron microscopy were harvested 3days post seeding, washed with PBS (3×3 ml), and fixed overnight at 4°C. in 2.5% (vol/vol) glutaraldehyde with 0.5% (wt/vol) tannic acid in0.07M sodium cacodylate buffer (pH 7.0). Following a rinse in 0.O1Mcacodylate-HCl buffer (pH 7.3), postfixation for 2 hours in 1% (wt/vol)aqueous osmium tetroxide at 4° C., lattices were dehydrated throughascending alcohols and cleared in propylene dioxide. Samples were theninfiltrated with propylene dioxide/araldite (1:1 vol/vol) for 1 hour,followed by 12 hours immersion in Araldite (trade mark) alone. Sampleswere then embedded in fresh Araldite, ultrathin sections cut andsequentially stained with saturated uranyl acetate followed by Reynoldslead citrate. See FIGS. 11A (control) and 11B (exposed to inhibitor).The arrow heads indicate the cellular attachments to the collagenmatrix. It is clear that the attachment of the fibroblasts to theirsurrounding collagen matrix was not affected by the inhibitor.

[0213] Discussion

[0214] These experiments showed that Galardin did not affect thecytoskeleton or cell matrix attachment of the fibroblasts. Effects onthe cytoskeleton or on cell matrix attachment were possible explanationsfor the effect of Galardin on contraction and therefore for the effectof MMP inhibitors on contraction.

[0215] General Discussion

[0216] A number of studies have suggested possible mechanisms for cellmediated collagen contraction. A number of workers have suggested thatMMPs are not produced or are produced during lattice contraction but arenot implicated in the contractile process. The above-describedexperiments demonstrate that cellular derived MMP activity is crucial tothe process of collagen contraction. The requirement of MMP activity forcontraction appears to be common to all of the fibroblasts tested.

[0217] The present invention is not to be limited in scope by theembodiments disclosed in the Examples which are intended to illustratethe invention. Indeed, various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art and are intended to fall within the scope ofthe appended claims.

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claims:
 1. The use of a matrix metalloproteinase inhibitor in themanufacture of a medicament for the treatment or prophylaxis of anatural or artificial tissue comprising extracellular matrix componentsto inhibit contraction of the tissue.
 2. The use of a collagenaseinhibitor in the manufacture of a medicament for the treatment orprophylaxis of tissue comprising collagen to inhibit contraction of thetissue resulting from contraction of the collagen.
 3. The use of amatrix metalloproteinase inhibitor to inhibit the invasion by cells,especially fibroblasts, into tissue comprising an extracellular matrixand/or the migration by cells, especially fibroblasts, in or throughtissue comprising an extracellular matrix by cells.
 4. A pharmaceuticalpreparation, other than a preparation suitable for use in the eye,suitable for application to a wound, burn or skin graft comprising amatrix metalloproteinase inhibitor.
 5. A pharmaceutical preparation asclaimed in claim 4 in the form of a solution, suspension, cream,ointment or gel in which the concentration of the matrixmetalloproteinase inhibitor is in the range of 0.4 μg/ml to 400 μg/ml.6. A method for the inhibition, for cosmetic reasons, of contraction oftissue comprising extracellular matrix components, which methodcomprises administering a matrix metalloproteinase inhibitor to thetissue.
 7. A method for the inhibition of contraction in vivo of anatural or artificial tissue comprising extracellular matrix components,which comprises administering a therapeutically effective amount of amatrix metalloproteinase inhibitor to the tissue.
 8. A method for theinhibition of contraction in vitro of a natural or artificial tissuecomprising extracellular matrix components, which comprisesadministering a matrix metalloproteinase inhibitor to the tissue.
 9. Amethod for the inhibition of contraction of tissue comprising collagenresulting from contraction of the collagen, which comprisesadministering a therapeutically effective amount of a collagenaseinhibitor to the tissue.
 10. The invention as claimed in any one ofclaims 1 to 9 in which the inhibitor is a broad spectrum matrixmetalloproteinase inhibitor.
 11. The invention as claimed in any one ofclaims 1 to 8 in which the inhibitor is capable of inhibiting one ormore of matrix metalloproteinases 1, 2 and
 3. 12. The invention asclaimed in claim 10 in which the inhibitor isN-(2(R)-2-(hydroxamido-carbonylmethyl)-4-methylpentanoyl]-L-tryptophanmethylamide.
 13. The invention as claimed in any one of claims 1 to 11in which the inhibitor is a peptide hydroxamic acid or apharmaceutically acceptable derivative thereof.
 14. The invention asclaimed in any one of claims 1 to 11 in which the inhibitor is an-agentwhich inhibits the production of matrix metalloproteinases.
 15. Theinvention as claimed in any one of claims to 11 in which the inhibitoris a monoclonal or a polyclonal antibody.
 16. The invention as claimedin any one of claims 1 to 15 in which more than one inhibitor is used,one inhibitor being a matrix metalloproteinase inhibitor and at leastone other inhibitor having inhibitory properties for an enzyme otherthan a matrix metalloproteinase.
 17. The invention as claimed in any oneof claims 1 to 16 in which more than one inhibitor is used, oneinhibitor being a collagenase inhibitor and at least one other inhibitorhaving inhibitory properties for a matrix metalloproteinase enzyme otherthan collagenase.
 18. The invention as claimed in any one of claims 1 to17 in which the inhibitor is used in combination with a cytokineinhibitor.
 19. The invention as claimed in any one of claims 1 to 18 inwhich the inhibitor is used in combination with a furtherpharmaceutically active ingredient chosen from the group of antibiotics,antifungals, steroids, enzyme inhibitors, growth promoters and healingpromoters.
 20. The invention as claimed in any one of claims 1, 2 and 6to 19 in which the contraction results from a pathological condition orfrom surgical or cosmetic treatment.
 21. The invention as claimed in anyone of claims 1 to 3 and 6 to 19 in which the tissue is an artificialskin.
 22. The invention as claimed in any one of claims 1 to 3 and 6 to20 in which the tissue is found in the eye.
 23. The invention as claimedin any one of claims 1, 2 and 4 to 20 in which the use is a prophylacticmeasure taken before any signs of contraction have been observed. 24.The invention as claimed in any one of claims 1 to 3 and 6 to 20 inwhich the contraction is associated with a chemical burn, a thermal burnor a radiation burn, a skin graft, a post-trauma condition resultingfrom surgery or an accident, glaucoma surgery, diabetes associated eyedisease, scleroderma, Dupytren's contracture, epidermolysis bullosa or ahand or foot tendon injury.
 25. A method of treating a human or othermammal to inhibit contraction of tissue comprising an extracellularmatrix component, especially contraction associated with a chemicalburn, a thermal burn or a radiation burn, a skin graft, a post-traumacondition resulting from surgery or an accident, glaucoma surgery,diabetes associated eye disease, scleroderma, Dupytren's contracture,epidermolysis bullosa or a hand or foot tendon injury, which comprisesadministering to the human or other mammal a therapeutically effectiveamount of a matrix metalloproteinase inhibitor.